The characterization of cellular gene expression finds application in a variety of disciplines, such as in the analysis of differential expression between different tissue types, different stages of cellular growth or between normal and diseased states. Recently, changes in gene expression have also been used to assess the activity of new drug candidates and to identify new targets for drug development. The latter objective is accomplished by correlating the expression of a gene or genes known to be affected by a particular drug with the expression profile of other genes of unknown function when exposed to that same drug. Genes of unknown function that exhibit the same pattern of regulation, or signature, in response to the drug are likely to represent novel targets for pharmaceutical development.
DNA arrays are particularly useful in gene expression analysis at the level of transcription (see, e.g., Ramsay, Nature Biotechnol. 16:40-44, 1998; Marshall and Hodgson, Nature Biotechnol. 16:27-31, 1998; Lashkari et al., Proc. Natl. Acad. Sci. (USA) 94:130-157, 1997; DeRisi et al., Science 278:680-6, 1997). In such analysis, the identity and abundance of a selected nucleic acid sequence in a sample is determined by measuring the level of hybridization of the nucleic acid sequence to probes on the DNA array that comprise complementary sequences. The selected nucleic acid sequence in a sample can be an mRNA, or a nucleic acid molecule derived from an mRNA that has a nucleic acid sequence that is identical to, or complementary to, all, or a portion, of the mRNA. Using DNA array expression assays, complex mixtures of labeled nucleic acids (e.g., mRNAs, or nucleic acid molecules derived from mRNAs) can be analyzed.
The nucleic acid molecules used to screen a DNA array should be representative of the mRNA population from which they are derived. All, or substantially all, of the sequences in the mRNA population should be represented in the nucleic acid molecule population used to screen the DNA array. For example, all portions of individual mRNA molecules should be equally represented in the nucleic acid molecule population used to screen the DNA array. In this regard, the use of oligo-dT primers, that hybridize to the polyA tail of mRNA molecules, to prime the enzymatic synthesis of complementary DNA molecules, results in the underrepresentation of the 3′ ends of long mRNA molecules in the population of complementary DNA molecules.
A proposed solution to this problem is to use a population of oligonucleotides, having random nucleic acid sequences, to prime the enzymatic synthesis of DNA molecules complementary to the template mRNA molecules. It is statistically likely that at least one of the random oligonucleotides will hybridize to at least one portion of each mRNA molecule in a population, thereby yielding a population of complementary DNA molecules that represent all, or substantially all, portions of all, or substantially all, mRNA molecules in the template population. A drawback to this approach, however, is that there is little or no amplification of the sequences in the template mRNA population, thereby limiting the practical usefulness of the technique, for example to produce enough probe to screen numerous DNA arrays.
Further, the nucleic acid molecules used to screen a DNA array should selectively hybridize to complementary nucleic acid molecules, and not hybridize, to a significant extent, to non-complementary nucleic acid molecules, immobilized on the DNA array. In this regard, the present inventors have observed that RNA molecules are typically more prone to hybridize to complementary nucleic acid molecules, immobilized on a DNA array, than are DNA molecules.
Thus, there is a need for methods for synthesizing DNA molecules from mRNA template molecules, wherein: (a) the synthesized DNA molecules represent all, or substantially all, portions of all, or substantially all, template mRNA molecules; (b) the abundance of each template mRNA molecule, and each portion of each template mRNA molecule, is identical, or substantially identical, to the abundance of the identical, or complementary, DNA sequence in the population of synthesized DNA molecules; and (c) the synthetic method is capable of amplifying a small amount of template mRNA (e.g., 1 μg or less) to yield sufficient probe to screen numerous DNA microarrays. Preferably, the synthesized DNA molecules selectively hybridize to complementary nucleic acid molecules, and do not hybridize, to a significant extent, to non-complementary nucleic acid molecules immobilized on a DNA array. Moreover, it is desirable that the synthetic methods controllably yield a population of DNA molecules wherein all, or substantially all, of the DNA molecules are complementary to either the sequences of the template mRNA molecules, or to the complementary sequences of the template mRNA molecules.
In one aspect, the present invention provides processed nucleic acid samples that meet the foregoing requirements, and methods for making such processed nucleic acid samples.